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(11) | EP 0 139 216 A2 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | Adult T cell leukemia virus antigen peptide |
| (57) Recombinant plasmids are constructed by inserting a DNA fragment coding for adult
T cell leukemia virus antigen peptide into a vector DNA. Microorganisms are transformed
with the recombinant plasmid and thereafter cultured to express the peptide. The adult
T cell leukemia virus antigen peptide is useful for the detection and diagnosis of
adult T cell leukemia. |
Background of the Invention
[0001] The present invention relates to an adult T cell leukemia virus antigen peptide, a recombinant plasmid bearing an inserted DNA fragment which codes for the peptide, a microorganism containing the recombinant plasmid, and a method of producing adult T cell leukemia virus antigen peptide using the microorganism.
[0002] Adult T cell leukemia virus (hereinafter referred to as ATLV), which is a synonym of human T cell leukemia virus (HTLV), is a retrovirus isolated from patients with adult T cell leukemia (hereinafter referred to as ATL) [Hinuma et al.: Proc. Natl. Acad. Sci., USA, 78, 6476-6480 (1981), Yoshida et al.: Proc. Natl. Acad. Sci., USA, 79, 2031-2035 (1982)]. There are numerous reports that ATL patients have a poor prognosis and that, efficacious treatment does not exist leading to a 50% mortality rate within 10 months.
[0003] In recent years, an antibody which reacts specifically with cultured MT-1 cells derived from ATL has been shown to exist in the serum of ATL patients (Hinuma et al., supra). The existence of this antibody has been confirmed subsequently in all ATL patients and the corresponding antigen is called ATL-associated antigen (hereinafter referred to as ATLA). It has been found that the antibody specific for ATLA (hereinafter referred to as Anti-ATLA antibody) exists in 25% of normal, healthy people in areas witn a high incidence of ATL. It has also been shown that the distribution of cases possessing the anti-ATLA antibody corresponds to the regions with high ATL incidence. Furthermore, it has been shown that the retrovirus is generated within MT-1 cells, that ATLA is mainly an antigen of this retrovirus and that anti-ATLA. antibody reacts with a structural protein of the virus, particularly the p24 protein. The existence of ATLV genome in the peripheral lymphocytes of patients has been established. ATLV has also been detected by culturing the lymphocytes of normal people who are positive to anti-ATLA antibody.
[0004] There is a very close correlation between ATL and ATLV, and ATLV is considered to be the causative virus of ATL. Though the route by which infection occurs is still unknown, it is considered that transfusion, maternal transmission and coitus transmission are the most likely routes. As 25% of healthy people in areas with a high incidence of ATL are anti-ATLA antigen positive, the likelihood of their being carriers of the disease is extremely high, which means that they must be avoided as blood transfusion donors.
[0005] Therefore early detection of the presence of anti-ATLA antibody will enable avoidance of transfusions from carriers and early ATL detection. At the present time, detection of anti-ATLA antibody is conducted using acetone fixed slides of MT-1 cells.
[0006] However, a more specific, simpler, faster method of anti-ATLA antibody detection and earlier ATL diagnosis are desirable. To this end, it has now been found that ATLV antigen peptide is accumulated and may be produced in good quantities by inserting the DNA fragment coding for the p24 structured protein within the gene for major antigen peptide in the ATLV genome, named the gag gene (group specific antigen), into a vector DTA by recombinant LNA technolog to prepare /prepare a recombinant DNA and then culturing microorganisms hosting the recombinant DNA.
[0007] The amino acid sequence from N-terminal to the 25th amino acid in p24 has previously been reported by Oroszlan et al.: Proc. Natl. Acad. Sci., USA, 79, 1291-1294 (1982). Furthermore, the entire DNA sequence of ATLV has been previously determined by the present inventors (European Patent Application No. 83-11 2261.9 filed December 7 , 1983). By the present invention, a process for the production of the ATLV antigen peptide in a microorganism is provided.
Summary of the Invention
[0008] In accordance with the present invention a recombinant plasmid is constructed by inserting a DNA fragment coding for adult T cell leuKemia virus antigen peptide in a vector DNA. The recombinant plasmid preferably includes a tryptophan promoter or a lactose promoter positioned upstream from the DNA fragment coding for adult T cell leuKemia virus antigen peptide. The present invention also provides a method for producing adult T cell leukemia virus antigen peptide by culturing, in a nutrient medium, a microorganism harboring a recombinant plasmid constructed by inserting a DNA fragment coding for adult T cell leukemia virus antigen peptide in a vector DNA plasmid., accumulating adult T cell leukemia virus antigen peptide in the culture and recovering the peptide therefrom. The invention further provides microorganism transformants harboring a recombinant plasmid containing a DNA fragment coding for adult T cell leukemia virus antigen peptide. Finally, according to the composition of matter aspects of the invention, adult T cell leukemia virus antigen peptide is provided.
Brief Description of the Drawings
[0009] In the accompanying drawings:
Fig. 1 illustrates the process for constructing plasmid pATK105 from plasmid pATK03;
Fig. 2 illustrates the process for constructing plasmids pTAG424A and pTAG424B from plasmid pATK105;
Fig. 3 illustrates the process for constructing plasmid pKYP-5;
Fig. 4 illustrates the process for constructing plasmid pKYP-10;
Fig. 5 illustrates tne process for constructing plasmid pKYP-100; and
Fig. 6 illustrates the process for constructing plasmid pTrS3.
Description of the Invention
[0010] The present invention provides an ATLV antigen peptide, a recombinant plasmid bearing an inserted DNA fragment coding for the peptide, a microorganism harboring the plasmid and a method for producing the ATLV antigen peptide using the microorganism.
[0011] The recombinant plasmid according to the invention can be constructed by insertion of a DNA coding for ATLV antigen peptide into a vector DNA using recombinant DNA techniques.
[0012] As structural proteins of the ATLV particle, pll, pl4, pl7, p24 and p45 are known. As the most commonly appearing among these is p24 and since it has a high reactivity with the serum of ATL patients, it is the most desirable as ATLV antigen peptide.
[0013] The plasmid pATK03 cloned by Seiki et al. [Seiki et al.: Proc. Natl. Acad. Sci., USA, 80, 3613-3622 (1983)] can be used as a source of the DNA coding for ATLV antigen peptide. The ATLV genome consists of the LTR at both ends and at least 3 genes, namely, gag, pol, and env. It is assumed that p24, whicn is the virus antigen about which the most detailed research is being conducted, is the product of the gag gene as it is a core protein. Actually the DNA sequence coding for the amino acid sequence determined by Gallo et al. [Oroszlan et al.: Proc. Natl. Acad. Sci., USA, 79, 1291-1294 (1982)] was detected in the gag gene. (Refer to the underlined section in Table 1 on page 15 and 16.) Thus, pATR03 is a clone which contains the entire gag gene
[0015] Any vector DNA can be utilized, provided it is autonomously replicable in the intended host microorganism and the inserted DNA coding for ATLV antigen peptide is expressed within a microorganism. For this expression, the respective DNA-sequence is, dependant from the host, operatively linked to an expression control sequence selected from the group of the E. coli λ promoter system, the E. coli lac system, the E. coli β-lactamase system, the E. coli trp-system, the E. coli lipoprotein promoter, yeast promoters and other encaryotic expression control sequences. It-is preferred to use a plasmid which includes a suitable promoter such as a tryptophan (trp) or lactose (lac) promoter downstream from which the subject DNA can be inserted. The downstream insertion site must be adjusted to have a suitable distance such as 6 - 18 base pairs, between the Shine-Dalgarno sequence (hereinafter referred to as SD sequence) and the translation initiation codon (ATG). One of the most suitable plasmids is pTrS3. Plasmid pTrS3 is constructed by the method described in Reference Example 3. Since pTrS3 vector has a distance (SD-ATG) of 13 base pairs between tne SD sequence and the translation initiator codon (ATG) downstream from the tryptophan promoter and a foreign DNA can be inserted immediately after ATG, any gene which has the frame conforming with the ATG will be expressed directly and efficiently using this vector.
[0016] Recomnination of a DNA coding for the ATLV antigen peptide such as the DNA coding for p24 from pATK03 and a vector DNA such as pTrS3 is generally carried out using recombinant DNA methods in which restriction enzymes are used to digest both DNAs followed by ligation using T4DNA ligase. Ligation may be conducted by a method employing fill-in reaction with DNA polymerase I Klenow fragment or a method using DNA linker. In the case of pATK03 and pTrS3, as snown in Figs. 1 and 2, an EcoRI-HindIII digestion fragment containing the gag gene from pATK03 is subcloned in pBR322. The recombinant plasmid pTAG424A is constructed by inserting a fragment obtained by Sau3A digestion of the subcloned pATK105 into the SphI site of pTrS3. In addition, pTAG424B can be obtained by re-ligation after cleavage of pTAG424A with SmaI and PvuII.
[0017] The reaction conditions necessary for the above-described preparation of the recombinant plasmid are generally as follows. DNA digestion with restriction enzymes is normally carried out by 15 minutes - 24 hours digestion of 0.1 - 100 µg of DNA, at 18 - 42°C, preferably 32 - 38°C, using 0.1 - 300 units, preferably 1 - 3 units, of restriction enzyme per 1 µg of DNA in 2 - 200 mM, preferably 10 - 40 mM Tris HC1 (pH 6.0 - 9.5, preferably pH 7.0 - 8.0), 1 - 15U mM NaCl and 2 - 20 mM, preferably 5 - 10 mM MgCl2. The reaction is terminated by heating at 55 - 75°C, preferably 63 - 70°C, for 5 - 30 minutes. The restriction enzymes may be inactivated by reagents such as phenol and dietnyl pyrocarbonate. Synthetic oligonucleotides are prepared by the diethyl phosphate method [H. G. Khorana et al.:. J. Mol. Biol., 72, 209 (1972)], the phosphotriester method [R. Crea et al.: Proc. Natl. Acad. Sci., USA, 75, 5765 (1978)] or the phosphite method [M. D. Matteucci et al.: J. Am. Chem. Soc. 103, 3185 (1981)].
[0018] Phosphorylation of the synthetic oligonucleotides is conducted at 20 - 40°C, preferably 35 - 38°C for 5 minutes to 2 hours, using 0.1 - 100 units of T4 polynucleotide kinase in 2 - 200 mM, preferably 10 - 70 mM Tris-HCl (pH 6.0 -9.5, preferably pH 7.0 - 8.0), 3 - 20 mM, preferably 4 - 10 mM MgCl2 and 1 - 10 mM dithiothreitol. Ligation of DNA fragments is conducted at 1 - 37°C, preferably 3 - 20°C, for 15 minutes to 72 hours, preferably 2 - 20 hours using 0.1 - 10 units of T4DNA ligase in 2 - 200 mM, preferaDly 10 - 70 mM Tris-HCl (pH 6.0 - 9.5, preferably pH 7.0 - 8.0), 2 - 20 mM, preferably 5 - 10 mM MgCl2, 0.1 - 10 mM, preferably 0.5 - 2 mM ATP and 1 - 50 mM, preferably 5 - 10 mM dithiothreitol.
[0019] Purification of the DNA fragments, recombinant plasmids, etc. is carried out by agarose gel electrophoresis.
[0020] ATLV antigen peptide is obtained by culturing a transformant obtained by introducing a recombinant plasmid such as pTAG424A or pTAG424B into a microorganism. It is desirable to use Escherichia coli as the host microorganism and HB101 or CSR 603 derived from the Eschericnia coli K-12 strain is preferably used. Transformation is carried out in accordance with the method of S. N. Cohen et al: Proc. Nat Acad. Sci., USA, 69, 2110 (1972). Transformants containing pTAG424A or pTAG424B can be screened and obtained as ampicillin-resistant strains. Culturing of transformants is carried out using a medium and culturing conditions selected to be suitable for the particular strain employed as the host microorganism. Culturing is continued until recoverable quantities of the peptide are expressed by the transformant. Thereafter the peptide is recovered by standard methods.
[0021] Expression of the ATLV antigen peptide by the transformant is detected in accordance with the Maxi-cell method as Sancar et al.: J. Bact. 137, 692-693 (1979). This involves culturing of the microorganism strain in M9-Casamino acid medium, followed by preferential chronosome activation using controlled ultra-violet irradiation. Tnis process therefore permits in consequence the predominant expression of genes which are present within tne plasmids, existing in multicopies. By using 35S-methionine as a marker, labelled proteins are detected by SDS polyacrylamide gel electrophoresis [Laemmli, Nature 227, 680 (1970)]. The polypeptide detected in this way is ascertained to give specific immune agglutination with patient serum, which indicates that it has the properties of ATLV antigen peptide. Isolation of the plasmids from the microorganisms is carried out in accordance with the method of H. C. Birnboim et al.: Nucleic Acids Research 7, 1513 (1979).
[0022] Certain specific embodiments of the invention are illustrated by the following representative examples.
Example 1
[0023] Insertion of the ATLV gag gene fragment into the expression vector pTrS3:
(1) Subcloning of the gag gene of plasmid pATK03 (Fig. 1): 600 µg of pATK03 were dissolved in 2 ml of a solution consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol and 100 mM NaCl. Then, 1,000 units of restriction enzyme ApaI (product of Boehringer Mannheim GmbH) were added and digestion was carried out at 37°C for 6 hours. DNA fragments were isolated by subjecting the digest to agarose gel electrophoresis. Hydroxyl apatite (product of Bio Rad Co., hereinafter referred to as HAP) was put into a groove formed directly in front of the desired fragment of 2.7 Kb on tne gel. Electrophoresis was continued and when the subject band was adsorbed on tne HAP, the DNA fragment-adsorbed HAP was collected with a pasteur pipette and put on a Sephadex G-50 column (1 cm x 20 cm) whicn had been equilibrated with 10 mM Tris-HCl (pH 7.5). DNA fragments were dissociated from the HAP with 0.5M EDTA (pH 8.0) and elution was continued with 10 mM Tris-HC1 (pH 7.5) to obtain the DNA fraction. After phenol and chloroform extraction of the fraction, a DNA fragment of 2.7 Kb was recovered by ethanol precipitation. Hereinafter, the method for recovery of a DNA fragment using agarose gel electrophoresis and HAP is referred to as AGE-HAP. Then, 40 ug of the DNA fragment were dissolved in 100 µℓ of a solution consisting of 20 mM Tris-HC1 (pH 7.5), 10 mM MgCl2 and 10 mM dithiothreitol. 10 units of restriction enzyme HaeII (product of Takcara Shuzo Co., the restriction enzymes hereinafter are all products of Takara Shuzo Co., unless otherwise specified) wese added and partial digestion of the DNA fragment was carried out at 37°C for 15 minutes. The-reaction solution was subjected to AGE-HAP and 5 µg of DNA fragment of 1,795 base pairs were obtained. Then, 5 µg of the DNA fragment were dissolved in a solution consisting of 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 and 1 mM dithiothreitol and 1 mM each of dATP, dTTP, dGTP and dCTP were added together with 15 units of Escherichia coli DNA polymerase I Klenow fragment (Bethesda Research LaDoratories Inc., hereinafter referred to as BRL). Fill-in reaction was performed at 15°C for 3 hours.
[0024] Separately, 4.8 µg of EcoRI linker (product of Takara Shuzo Co.) were dissolved in 30 µℓ of a solution of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM dithiothreitol and 1 mM ATP. Five units of T4 polynucleotide kinase (product of Takara Shuzo ·Co. ) were added and the mixture was subjected to phosphorylation reaction. Then, 2.4 µg of phospnorylated EcoRI linker were mixed with 5 µg of the fragment partially digested with HaeII described above. The mixture was then dissolved in 50 µℓ, of a solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 1 mM ATP, followed by addition of 2.5 units of T4DNA ligase (product of Taxara Shuzo Co.). After ligation at 4°C for 16 hours, the whole DNA was recovered by ethanol precipitation.
[0025] Then, 4 µg of DNA fragment with attached EcoRI linker were dissolved in 100 µℓ of a solution consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 60 mM NaCl. Five units each of EcoRI and HindIII were added and digestion reaction was carried out at 37°C for 2 hours. From this digest, 1.5 µg of EcoRI-HindIII digested fragment of 1,453 base pairs were obtained by AGE-HAP.
[0026] Separately, 5 µg of pBR322 (4.4 Kb) [Bolivar et al.: Gene, 2, 95 (1977)] were dissolved in 100 µℓ of a solution consisting of 20 mM Tris-HCl, 10 mM MgCl2 and 10 mM dithiothreitol. Then, 5 units each of EcoRI and HindIII were added and digestion was carried out at 37°C for 2 hours. 2.5 µg of EcoRI-HindIII fragment of about 4.3 Kb wererecovered by AGE-HAP.
[0027] 0.2 µg of the fragment and 0.35 µg of the EcoRI-HindIII fragment of 1,453 base pairs from pATK03 were dissolved in 50 µℓ, of a solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2 10 mM dithiothreitol and 1 mM ATP. Then, 2.5 units of T4DNA ligase were added and ligation reaction was carried out at 4°C for 16 hours.
[0028] Using this ligation solution, Escherichia coli K-12, HB101 strain [Bolivar et al.: Gene 2, 75 (1977)] was transformed by conventional technique and an ampicillin-resistant (ApR) strain was obtained. The recombinant plasmid pATK105 shown in Fig. 1 was isolated from the strain by conventional technique. The structure of pATK105 was determined by AGE after digestion with the restriction endonucleases, EcoRI, HindIII and PstI. Escherichia coli K-12, HB101 strain containing plasmid pATK105 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (Fermentation Research Institute) as Escherichia coli EATK105, FERM BP-340.
[0029] pATK105 is a plasmid containing a sequence extending from the 18th base pair upstream from the ATG codon to the 139th base pair downstream from the terminator codon of the ATLV gag gene and is thus a suitable source of the gag gene for recombination.
[0031] A DNA fragment containing the p24 coding region was cleaved from pATK105 and ligated directly next to the ATG codon of the expression vector pTrS3 in the following manner.
[0032] 40 µg of pATK105 were dissolved in 200 µℓ of a solution consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol and 50 mM NaCl. Then, 50 units of Sau3A were added and digestion was carried out at 37°C for 3 hours. 25 µg of Sau3A DNA fragment of 1,022 base pairs were obtained from this reaction solution by AGE-HAP. Then, about 2 µg of the DNA fragment were dissolved in 30 µℓ of a solution consisting of 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2 and 1 mM dithiothreitol, followed by addition of 1 mM each of dATP, dTTP, dGTP and dCTP, together with 3 units of Escherichia coli DNA polymerase I · Klenow fragment. Fill-in reaction was carried out at 15°C for 3 hours.
[0033] Separately, 10 µg of pTrS3 (3.8 Kb) obtained by the reference example method were dissolved in 100 µℓ of a solution consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol and 150 mM NaCl. Then, 10 units of SphI (product of BRL) were added and digestion reaction was carried out at 37°C for 3 hours. The DNA recovered by ethanol precipitation was dissolved in 100 µℓ of a solution consisting of 50 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 10 mM dithiothreitol and 1 mM each of dATP, dTTP, dGTP and dCTP. Then, 10 units of Escherichia coli DNA polymerase I . Klenow fragment were added and fill-in reaction was carried out at 15°C for 3 hours. About 1 µg of a DNA fragment of 3.8 Kb was recovered by AGE-HAP.
[0034] Thereafter, 0.1 µg of the DNA fragment and 0.4 µg of the Sau3A DNA fragment prepared by the above-described fill-in reaction were dissolved in 50 µℓ, of a solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 1 mM ATP. 2.5 units of T4DNA ligase were added and ligation reaction was carried out at 4°C for 16 hours. Escherichia coli K-12, HB101 strain was transformed by conventional technique using this ligation solution and a ApR strain was obtained. Plasmids were isolated from the strain by conventional technique and the recombinant plasmid pTAG424A was recovered. The structure of pTAG424A was confirmed by AGE after digestion with Xhol, PstI, PvuII, Sau3A, Clal, SmaI and BamHI. The structure between SD-ATG was investigated by the method of Maxam and Gilbert, Proc. Natl. Acad. Sci., USA, 74, 560 (1977). It was thus determined that pTAG424A codes for a polypeptide consisting of a total of 317 amino acids, wherein 17 amino acids and 85 amino acids derived from the gag gene are respectively attacned to the N-terminal .and C-terminal of p24. It was also confirmed that the base sequence between SD-ATG is
[0035] Escherichia coli K-12, HB101 strain which contains plasmid pTAG424A has been deposited with the Fermentation Research Institute as Escherichia coli ETAG424A, FERM BP-341. A derivative can be constructed from pTAG424A by removing a DNA sequence whicn corresponds to an amino acid sequence other than p24 using appropriate restriction enzymes, exonuclease BAL31 and Escherichia coli DNA polymerase I.
Example 2
[0036] Construction of pTAG424B from pTAG424A: To contruct plasmid pTAG424B, 5 µg of pTAG424A (about 4.8 Kb) obtained in Example 1 were dissolved in a solution consisting of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 50 mM NaCl. Then, 10 units each of SmaI and PvuII were added and digestion reaction was carried out at 37°C for 3 hours. From this reaction solution, 2 µg of Smal-and PvuII-cleaved DNA fragment of 3.1 Kb were recovered by AGE-HAP. Then, 0.2 µg of the DNA fragment were dissolved in 50 µℓ of a solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 1 mM ATP. After addition of 2.5 units of T4DNA ligase, ligation reaction was carried out at 4°C for 16 hours. The reaction solution was used to transform Escherichia coli K-12, HB101 strain by conventional technique to obtain an ApR strain. Plasmids were isolated from this strain by conventional technique to obtain the recombinant plasmid, pTAG424B, shown in Fig. 2. The structure of pTAG424B.was confirmed by AGE after digestion with BamHI, PstI, SmaI and PvuII. Tne polypeptide encoded by pTAG424B compared with that encoded by pTAG424A is shorter by a section corresponding to 52 amino acids at the C-terminal and has a section corresponding to 6 amino acids derived from pBR322.
[0037] Escherichia coli K-12, HBl01 strain containing plasmid pTAG424B has been deposited with the Fermentation Research Institute as Escnerichia coli ETAG424B, FERM BP-342.
Example 3
[0038] Identification of the peptides encoded by pTAG424A and pTAG424B was carried out by the Maxi-cell method of Sancar et al.: J. Bact., 137, 692-693 (1979) as described below.
[0039] pTAG424A and pTAG424B, respectively, were used to transform Escherichia coli CSR603 strain (recAl, uvrA6, phr-1) by conventional technique. ApR strains, namely a strain containing pTAG424A and a strain containing pTAG424B, were obtained. These strains and also the strain containing pTrS3 (FERM P-6735) were inoculated in 10 mℓ of an M9-Casamino acid medium (pH 7.4) consisting of 0.6% Na2HPO4, 0.3% KH2P04, 0.05% NaCl, 0.1% NH4Cl, 2 mM MgSO4, 0.2% glucose, 0.1 mM CaCl2 and 0.5% Casamino acid and further containing 50 µg/mℓ ampicillin (Ap) and 50 µg/mℓ tryptophan. Culturing was carried out with shaκing at 37°C. At OD660 0.2, the culture fluid was transferred to a watch glass containing a stirring bar. Agitation was continued with the stirrer during 10 seconds of irradiation at 90 cm directly under a UV lamp (15W). The culture fluid was then transferred to a large test tube (50 mℓ) and snaking was continued at 37°C for 1 hour. Thereafter, 100 µg/mℓ (final concentration) D-cycloserine were added and culturing was carried out at 37°C for 16 hours. The culture fluid was then centrifuged (3300 rpm, 10 minutes) and cells were harvested. The cells were washed twice with sulfur-free M9 medium (M9-Casamino acid medium containing 2 mM MgCl2 from which Casamino acid and MgSO4, were removed). The cells were then suspended in 1 mℓ of sulfur-free M9 medium containing 50 µg/mℓ Ap and 10 µg/mℓ indole acrylic acid (IAA), and the suspension was shaken at 37°C for 1 hour. 35S-methionine (~1000 Ci/m mole, product of Amersham Co.)
[0040] was added to give 20 µCi/mℓ and culturing was carried out with shaking at 37°C for 1 hour to incorporate the marker. The cells were collected by centrifugation (3,300 rpm, 10 minutes) and suspended in 100 µℓ of Laemmli's sample buffer [Laemmli, Nature, 227, 680 (1970)]. The suspension was then heated at 100°C for 5 minutes to lyse the cells. Then, 20 µℓ of the lysate were subjected to SDS-polyacrylamide gel electrophoresis using the above referenced method of Laemmli.
[0041] Following the electrophoresis, the 35S-labeled peptide was detected by fluorography (EN3 HANCE, product of New England Nuclear Co.). As a result, only a mature β-lactamase band was detected in the pTrS3 vector. However, in pTAG424A, a peptide band with a molecular weight of about 35,000, and in pTAG424B, a peptide band with a molecular weight of about 30,000, was detected in addition to the β-lactamase band. The molecular weights exhibited by the pTAG424A and pTAG424B bands are almost identical to the molecular weights of the polypeptides estimated from the base sequence of the gag gene DNA fragment.
Example 4
[0042] In this example, the cells labeled in Example 3 were solubilized and extracts were subjected to immunoprecipitation reaction with the serum of ATL patients and healthy people by the method of Richert et al.: J. Virol. 31, 695-706 (1979). By analyzing the sediment by SDS-polyacrylamide gel, it was determined that the peptides encoded by pTAG424A and pTAG424B do not react at all with the serum of healthy people but react specifically with the serum of ATL patients. This indicates that the peptides encoded by pTAG424A and pTAG424B have the properties of ATLV antigen and are suitable for the detection of anti-ATL antibody.
Reference Example 1
[0043] Construction of a plasmid vector pKYPlO having a trp promoter:
(a) Purification of tryptophan transducing phage DNA:
A λtrp phage, λcI857trpEDlO (referred to as λtrp ED hereinafter) [G. F. Miozzari et al.: J. Bacteriol. 133, 1457 (1978)] was lysogenized in Escherichia coli JA 194 strain (F , λ, r , m , AtrpE5, leu6) [J. Carbon et al.: Recombinant Molecules p.355 (1977), Raven Press]. The resultant lysogenic strain, JA 194 (trpED) was cultured at 42°C for 30 minutes to induce XtrpED phages and prepare Xphage lysate. The λphages were purified from the Xphage lysate by the cesium chloride equilibrium density gradient centrifugation method of Yamakawa et al: "Chemicals of Nucleic Acids I", Tokyo Kagaku Dojin pp.54 - 61 (1974). The λpnages were further purified by phenol treatment and chloroform treatment according to the method of Yamakawa et al.: "Chemicals of Nucleic Acids I" Tokyo Kagaku Dojin, p.62 - 65 (1974).
(b) Cloning of the trp promoter into a plasmid:
To clone the trp operon from λtrpED phage DNA, 8 µg of XtrpED DNA were digested at 37°C for 2 hours with 16 units of EcoRI and 16 units of HindIII in 20 mM Tris-HCl (pH 7.5), 75 mM NaCl, 10 mM MgCl2 and 5 mM dithiothreitol. Separately, 1 µg of plasmid pBR325 DNA was digested with 2 units of EcoRI and 2 units of HindIII by the same method as mentioned above (final volume: 30 µl). The reactions were stopped by heating at 65°C for 5 minutes. Then, 15 µℓ each of the digests were mixed and 500 µM (final concentration) ATP and 5 units of T4DNA ligase were added. The mixture was allowed to react at 4°C for 18 hours. Escherichia coli C600 SF strain, [Cameron et al.: Proc. Natl. Acad. Sci. 72, 3416 (1975)] was transformed with the obtained plasmid mixture by the method of S. N. Cohen et al.: Proc. Natl. Acad. Sci. 69, 2110 (1972) and transformants which were resistant to ampicillin (ApR), resistant to tetracycline (Tc ) and sensitive to chloramphenicol (CmS) were selected. Plasmid DNAs were - isolated from the transformants of Escherichia coli. One of the plasmid DNAs was named pKYP-l. This plasmid was digested with TaqI and EcoRI and the digest was subjected to purification by agarose gel electrophoresis to obtain a 2.6 Kb DNA fragment containing the tryptophan promoter and SD sequence. The 2.6 Kb DNA fragment was cloned in a known vector, pBR322, by the method as illustrated in Fig. 3. That is, 8 pg of.pBR322 were digested at 45°C for 60 minutes with 2 units of TaqI in 100 µℓ of a reaction mixture comprising 10 mM Tris-HCl (pH 8.4), 6 mM MgCl2, 100 mM NaCl and 6 mM 2-mercaptoethanol. After partial digestion with TaqI, the digest was subjected to low-gelling-temperature agarose gel electrophoresis [Lars Wieslander: Analytical Biochemistry 98, 305 (1979)] to obtain a purified 4.36 Kb DNA fragment. About 1.5 µg of the DNA fragment were completely digested at 37°C for 3 hours with 3 units of EcoRI. About 1.0 µg of an about 4.33 kb DNA fragment was recovered by low-gelling-temperature agarose gel electrophoresis in a similar manner as above. Then, 12 µg of pKYP-1 DNA were partially digested with 3 units of TaqI. The digest was subjected to low-gelling-temperature agarose gel electrophoresis to obtain about 2 µg of a purified 8.5 Kb DNA fragment and the DNA was then completely digested with EcoRI by the same procedure as above to obtain about 0.5 µg of a purified 2.6 Kb DNA fragment. Then, 0.4 µg of the 4.33 Kb DNA of pBR322 and 0.25 µg of the 2.6 Kb DNA fragment of pKYP-1 thus obtained were added to 20 µℓ of a reaction solution consisting of 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2 and 10 mM dithiothreitol. Then, 0.5 mM ATP and 4 units of T4DNA ligase were added to the mixture and reaction was carried out at 4°C for 18 hours. Escherichia coli C600 SF8 strain was transformed with the recombinant plasmid DNA obtained as above. The plasmids in the transformant having ApR and TcR were isolated. The plasmid DNA was digested with 6 restriction endonucleases, EcoRI, HindII, ClaI (product of Boehringer Mannheim GmbH), HpaI, HincII and BamHI to analyze the structure of the plasmid. The plasmid was named pKYP-5.
(c) Preparation of a portable promoter from the trp promoter:
The plasmid vector pKYP-5 is applicable as a DNA introducing vector since it has a ClaI site and a HindIII site on the DNA of 1 to 20 base pairs downstream from the SD sequence. However, another Clal site is present in pKYP-5 DNA besides the Clal site immediately after the SD sequence and the fragment with the trp promoter obtained by cutting pKYP-5 DNA with EcoRI and HindIII is a little too large, i.e. 2.65 Kb. For convenience of use, pKYP-5 was improved by the process as illustrated in Fig. 4 to obtain a plasmid having a DNA fragment containing a
shorter tryptophan promoter. That is, pKYP-5 DNA was digested with HpaII and HindIII and the digest was purified to obtain a DNA fragment of about 340 bp (base pairs). The fragment was inserted into the pBR322 digested with ClaI and HindIII as illustrated in Fig. 4 to obtain pKYP-10. The structure of pKYP-10 was determined by agarose gel electrophoresis after digestion with EcoRI, ClaI, HindIII and HpaI.Reference Example 2
[0044] Construction of pKYP100 (refer to Fig. 5): To construct plasmid pKYP100, 50 µg of pKYPlO were digested with 50 units of HhaI in 100 µℓ of a reaction solution containing 10 mM Tris-HC1 (pH 7.5), 7 mM MgCl2 and 6 mM 2-mercaptoethanol at 37°C for 2 hours. After digestion with HhaI, a DNA fragment of about 180 bp containing the trp promoter was purified by 5% polyacrylamide gel electrophoresis [A. M. Maxam et al.: Proc. Natl. Acad. Sci. 74, 560 (1977), referred.to as PAGE hereinafter]. In the purification step, two DNA fragments other than the desired DNA were obtained because of incomplete purification by PAGE. The three purified DNA fragments (total amount: about 4 µg) were allowed to react with 8 units of Escherichia coli DNA polymerase I Klenow fragment in 30 µℓ of a reaction solution containing 50 mM Tris-HCl (pH 7.6), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP and 0.25 mM dTTP at 15°C for 2 hours. By the reaction, the 3'-protruding end formed by HhaI digestion was changed to flush end by the 3'→5' exonuclease activity and 5' → 3' repairing synthesis activity of DNA polymerase I Klenow fragment. Subsequently, DNA polymerase I Klenow fragment was inactivated by heating at 72°C for 30 minutes and the NaCl concentration was adjusted to 50 mM with 1 M NaCl. 8 units of HindIII were added and the mixture was allowed to react at 37°C for 2 hours. After digestion with HindIII, the DNA fragment of about 100 bp containing the trp promoter was isolated and purified by PAGE.
[0045] Separately, 5 µg of plasmid pBR322 were digested with 8 units of EcoRI in 20 µℓ of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 7 mM MgCl2 and 6 mM 2-mercaptoethanol at 37°C for 2 hours. After phenol and chloroform extraction and ethanol precipitation, the precipitated DNA fragment was dissolved in 20 µℓ of a mixture of 50 mM Tris-HCl (pH 7.6), 7 mM MgCl2, 6 mM 2-mercaptoethanol, U.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP and 0.25 mM dTTP. Then, 8 units of Escherichia coli DNA polymerase I · Klenow fragment were added and the mixture was allowed to react at 15°C for 2 hours. The 5'-protruding end formed by EcoRI digestion was changed to flush end by tne repairing synthesis activity of DNA polymerase I · Klenow fragment. The DNA polymerase I Klenow fragment was inactivated by heating at 72°C for 30 minutes and the NaCl concentration was adjusted to 50 mM with 1 M NaCl. 8 units of HindIII were added and the mixture was allowed to react at 37°C for 2 hours. After digestion with HindIII, the larger plasmid DNA fragment of about 4.33 Kb was purified by low-gelling-temperature agarose gel electrophoresis.
[0046] About 50 ng of the DNA fragment 3 of about 100 bp containing the trp promoter and obtained above, about 0.2 µg of the DNA fragment 6 of about 4.33 Kb derived from pBR322 and obtained above, and 50 ng of 5'-phosphorylated Xhol linker (pCCTCGAGG, product of Collaborative Research) were ligated with 1 unit of T4DNA ligase in 20 µℓ of a reaction solution containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 0.5 mM ATP at 4°C for 40 hours. Escherichia coli HB101 was transformed with the thus obtained recombinant plasmid DNA and plasmid DNAs were isolated and purified from the ApRTcR transformants. These plasmid DNAs were digested with restriction enzymes EcoRI, XhoI, HindIII, HaeIII, ClaI, TaqI (product of BRL) and RsaI (product of New England Biolabs) to select the plasmid 7 wherein the DNA fragment 3 of about 100 bp containing the trp promoter and XhoI linker 4 were cloned. This plasmid was named pKYPlOO.
Reference Example 3
[0047] Construction of plasmid vector pTrS3 bearing the initiation codon for translation ATG and SphI cleavage site downstream from the trp promoter and the ribosome. binding site (refer to Fig. 6):
Plasmid pTrS3 is obtained from pKYPlOO constructed as in Reference Example 2 in the following manner. 5 µg of pKYP100 8 were allowed to react with 5 units of ClaI in 20 µℓ of a reaction solution containing 10 mM Tris-HCl (pH 7.5), 7 mM MsgCl2 and 6 mM 2-mercaptoethanol at 37°C for 2 hours. The reaction was stopped by heating at 65°C for 5 minutes. Then, 2 µℓ of 100 mM Tris-HCI (pH 7.5), 70 mM MgCl2, 1.0 M NaCl, 60 mM 2-mercaptoethanol, 16 µℓ of distilled water and 7 units of SphI (product of Boehringer Mannheim GmbH) were added and the mixture was allowed to react at 37°C for 2 hours. The reaction was stopped by heating at 65°C for 5 minutes and the larger plasmid DNA fragment 9 (about 3.82 Kb) was purified by low-gelling-temperature agarose gel electrophoresis.
[0048] Separately, two species of oligonucleotides, 5'-CGATAAGCTATGCATG-3' and 5'-CATAGCTTAT-3' were synthesized by the phosphotriester method. The two synthesized oligonucleotides were 51-phosphorylated and 20 µM each of the oligonucleotides were mixed with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA. The mixture was incubated at 65°C for 10 minutes, at 37°C for 120 minutes and at room temperature for 120 minutes to anneal them. The two DNA chains were annealed as illustrated below.
pCGATAAGCTATGCATG
TATTCGATACp
[0049] Both ends of the resulting DNA fragment can be ligated with the DNA fragment having sticky ends formed by digestion with ClaI or SphI and the ligated DNA has a ClaI cleavage site or SphI cleavage site for reconstruction. The annealed DNA 10 of the two oligonucleotides and the plasmid DNA fragment 9 purified as above were mixed and ligated with T4DNA ligase. That is, 1 pM each of the two oligonucleotides, pCGATAAGCTATGCATG and pCATAGCTTAT were annealed and about 0.15 µg of the purified plasmid DNA fragment 9 was added. Then, 0.5 unit of T4DNA ligase were added and the mixture was allowed to react in 20 µℓ of a reaction solution containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 10 mM dithiothreitol and 0.5 mM ATP at 4°C for 16 hours.
[0050] Escherichia coli HB101 was transformed with the resulting recombinant plasmid DNA. Plasmid DNAs were isolated from tne thus obtained transformant resistant to ampicillin and sensitive to tetracycline (ApRTcS) and purified. These plasmid DNAs were digested with EcoRI, XhoI, PstI, ClaI and SphI to recognize the formation of the desired plasmid vector pTrS3 11. It was recognized by the method of Maxam and Gilbert, Proc. Natl. Acad. Sci. 74, 560 (1977) that the base sequence of the DNA between the ClaI site and SphI site of pTrS3 was ATCGATAAGCTATGCATGC. Escherichia coli containing pTrS3 was deposited with the Fermentation Research Institute on September 29, 1982 as Escherichia coli ITrS-3 FERM P-6375. The deposit was transferred to the international deposit on July 26, 1983 and assigned accession number FERM BP-328.